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- EMDB-20567: The axonemal doublet microtubule focusing on the I1 dynein region... -

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Basic information

Entry
Database: EMDB / ID: EMD-20567
TitleThe axonemal doublet microtubule focusing on the I1 dynein region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas ida7-1 mutant cilia
Map dataThe I1 dynein region of the Chlamydomonas ida7-1 mutant
Sample
  • Organelle or cellular component: The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsFu G / Nicastro D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM083122 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM112050 United States
CitationJournal: FASEB J / Year: 2021
Title: Structural organization of the intermediate and light chain complex of Chlamydomonas ciliary I1 dynein.
Authors: Gang Fu / Chasity Scarbrough / Kangkang Song / Nhan Phan / Maureen Wirschell / Daniela Nicastro /
Abstract: Axonemal I1 dynein (dynein f) is the largest inner dynein arm in cilia and a key regulator of ciliary beating. It consists of two dynein heavy chains, and an intermediate chain/light chain (ICLC) ...Axonemal I1 dynein (dynein f) is the largest inner dynein arm in cilia and a key regulator of ciliary beating. It consists of two dynein heavy chains, and an intermediate chain/light chain (ICLC) complex. However, the structural organization of the nine ICLC subunits remains largely unknown. Here, we used biochemical and genetic approaches, and cryo-electron tomography imaging in Chlamydomonas to dissect the molecular architecture of the I1 dynein ICLC complex. Using a strain expressing SNAP-tagged IC140, tomography revealed the location of the IC140 N-terminus at the proximal apex of the ICLC structure. Mass spectrometry of a tctex2b mutant showed that TCTEX2B dynein light chain is required for the stable assembly of TCTEX1 and inner dynein arm interacting proteins IC97 and FAP120. The structural defects observed in tctex2b located these 4 subunits in the center and bottom regions of the ICLC structure, which overlaps with the location of the IC138 regulatory subcomplex, which contains IC138, IC97, FAP120, and LC7b. These results reveal the three-dimensional organization of the native ICLC complex and indicate potential protein-protein interactions that are involved in the pathway by which I1 regulates ciliary motility.
History
DepositionAug 7, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseAug 12, 2020-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 128
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20567.png

Downloads & links

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Map

FileDownload / File: emd_20567.map.gz / Format: CCP4 / Size: 469.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe I1 dynein region of the Chlamydomonas ida7-1 mutant
Voxel sizeX=Y=Z: 10.77 Å
Density
Contour LevelBy AUTHOR: 128.0 / Movie #1: 128
Minimum - Maximum86.202286 - 172.44293
Average (Standard dev.)121.9728 (±9.341913)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-3-37
Dimensions406050
Spacing604050
CellA: 646.2 Å / B: 430.80002 Å / C: 538.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.7710.7710.77
M x/y/z604050
origin x/y/z0.0000.0000.000
length x/y/z646.200430.800538.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-3-30-37
NC/NR/NS604050
D min/max/mean86.202172.443121.973

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Supplemental data

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Sample components

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Entire : The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia

EntireName: The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia
Components
  • Organelle or cellular component: The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia

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Supramolecule #1: The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia

SupramoleculeName: The I1 dynein averaged from Chlamydomonas ida7-1 mutant cilia
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: back-side blotting with No.1 Whitman filter for 1.5-2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 8.0 µm / Calibrated defocus min: 6.0 µm / Calibrated magnification: 13500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF 2000 / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 9 / Number images used: 1051 / Software - Name: MATLAB
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD / Number subtomograms used: 1051

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